EP2697384B1 - Method for producing butyric acid, butanol and butyrate ester - Google Patents

Method for producing butyric acid, butanol and butyrate ester Download PDF

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EP2697384B1
EP2697384B1 EP12771147.1A EP12771147A EP2697384B1 EP 2697384 B1 EP2697384 B1 EP 2697384B1 EP 12771147 A EP12771147 A EP 12771147A EP 2697384 B1 EP2697384 B1 EP 2697384B1
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fermentation
lactic acid
acid
carbohydrate
feedstock
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French (fr)
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EP2697384A1 (en
EP2697384A4 (en
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Yun-Huin Lin
Hom-Ti Lee
Hsiu-Yin YIN
Sz-Chwun John Hwang
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Industrial Technology Research Institute ITRI
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/52—Propionic acid; Butyric acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • Novel methods for producing butyric acid, butanol and butyrate ester are disclosed herein.
  • the method uses lactic acid and at least one carbohydrate as a feedstock for fermentation to produce butyric acid and butanol with a higher carbon yield than conventional fermentation methods using only carbohydrates as a carbon source.
  • Acetone-butanol-ethanol fermentation is an anaerobic process that uses bacterial fermentation to produce acetone, butanol and ethanol from carbohydrate.
  • the process also usually uses a strain of bacteria from the Clostridia Class. Clostridium acetobutylicum is the most well known strain, and Clostridium beijerinckii has also been used.
  • butyric acid and acetic acid are produced, and the culture then undergoes a metabolic shift and solvents, such as butanol, acetone and ethanol, are formed.
  • the process produces these solvents in a ratio of 3-6-1, or 3 parts acetone, 6 parts butanol and 1 part ethanol.
  • the fermentation processes are generally limited by low product yield, low productivity, and low product concentration.
  • the low product yield is due to the low conversion rate of substrate to product as a result of the production of CO 2 during the fermentation process.
  • the volatile organic compounds that are manufactured during fermentation process include ethanol or butanol. Additionally, large amounts of carbon dioxide are produced, generally 40-50%, and therefore these solvents are called "half-burn fuel.”
  • U.S. Patent No. 7,455,997 describes a two-step fermentation process using plant-derived feedstock including two polysaccharides, a first one which is more readily hydrolysable and a second one which is more difficult to hydrolyze.
  • the two polysaccharides are hydrolyzed by an acid and by an enzyme sequentially during the process to generate a mixture of fermentation products including, e.g., ethanol, glycerol, acetone, n-butanol, butanediol, isopropanol, butyric acid, methane, citric acid, fumaric acid, lactic acid, propionic acid, etc.
  • 5,132,217 teaches using nutrients selected from fructose, glucose, glycerol and sucrose as the main carbon source for fermentation in the presence of Clostridium to produce butyric acid.
  • the carbon yield is 34% from glucose and 33% from sucrose according to the examples.
  • Another process is described in US 2008/0248540 and WO 2008/124490 , which provide a yield of up to 48% (g/g), the theoretical conversion rate from glucose to butyric acid.
  • the same publications also disclose a two-step process to convert glucose to butanol.
  • the present disclosure provides methods for producing butyric acid and butanol with improved carbon conversion and carbon yield compared to conventional carbohydrate fermentation.
  • Novel methods are disclosed for producing butyric acid by fermenting a feedstock using a butyric acid-producing bacterium, wherein the feedstock comprises lactic acid and at least one carbohydrate.
  • the present disclosure is directed to methods of producing butyric acid, comprising fermenting a feedstock with at least one butyric acid-producing bacterium to produce butyric acid, wherein the feedstock comprises lactic acid and at least one carbohydrate.
  • the at least one butyric acid-producing bacterium comprises at least one strain of Clostridium.
  • the at least one strain of Clostridium is chosen from C . tyrobutyricum, C. butyricum and C. beijerinckii. In another embodiment, the at least one strain of Clostridium is C . tyrobutyricum.
  • the at least one carbohydrate is chosen from monosaccharides, disaccharides, polysaccharides, and mixtures thereof. In some embodiments, the at least one carbohydrate is chosen from monosaccharides and disaccharides.
  • the monosaccharide is glucose, xylose, galactose or a mixture thereof.
  • the disaccharide is lactose, sucrose, cellobiose, or a mixture thereof.
  • the polysaccharide is chosen from starch, glycogen, cellulose, and a mixture thereof.
  • the at least one carbohydrate comprises at least one soluble carbohydrate obtained from treating biomass with an enzymatic saccharification process.
  • the weight ratio of lactic acid and the at least one carbohydrate in the feedstock ranges from about 1 to about 10, from about 1 to about 3, or from about 1 to about 1.5.
  • the at least one strain of Clostridium is C . tyrobutyricum
  • the feedstock comprises lactic acid and glucose
  • the fermentation method provides a butyric carbon yield higher than that of a conventional fermentation using carbohydrate(s) as the only substrate.
  • the method provides a butyric carbon yield higher than about 66%, such as a butyric carbon yield higher than 69%.
  • the methods of the present disclosure generate less CO 2 compared to a conventional fermentation using carbohydrates(s) as the only substrate.
  • the lactic acid in the feedstock is converted to butyric acid without generation of CO 2 .
  • the conversion to butyric acid results in no detectable level of CO 2 .
  • the fermenting step continues for a time chosen from when a substantial amount of the feedstock is consumed, and when no substantial increase in butyric acid is observed. For example, the fermenting step continues for about 15 to about 80 hours, such as for about 30 to about 75 hours.
  • lactic acid and at least one carbohydrate may be sequential to the feedstock in any order, before or after the fermenting step commences.
  • the fermentation method further comprises hydrogenating the butyric acid to produce butanol by chemical or biochemical processes.
  • the hydrogenating step can be conducted using chemical hydrogenation, such as catalytic hydrogenation or transfer hydrogenation.
  • the hydrogenating step uses at least one butanol-producing microorganism.
  • the microorganism of the at least one butanol-producing microorganism is a solventogenesis phase of a Clostridium strain, such as C . acetobutylicum, C. beijerinckii, C. aurantibutyricum, and C . tetanomorphum.
  • the fermentation method further comprises esterifying the butyric acid in the presence of a catalyst and an alcohol to produce a butyrate ester. In some other embodiments, the butyrate ester may be further reduced to obtain butanol.
  • lactic acid such as in lactic acid and butyric acid
  • lactic acid may include: free lactic acid, sodium lactate, ammonium lactate, lithium lactate, calcium lactate, potassium lactate, magnesium lactate, ammonium lactate, and aluminum lactate.
  • butyric acid and “butyrate” are interchangeable to describe the acid or its deprotonated form, depending on the pH value of its environment. The same applies to other acids, for example, lactic acid/lactate, propionic acid/ propionate, and acetic acid/acetate.
  • chemical names described herein refer to all isomeric forms of the chemical names, such as enantiomers, diastereomers, and conformational isomers.
  • lactic acid “glucose,” “xylose,” “galactose” refers to both D and L isomers.
  • carbohydrate is capable of existing in both open-chain and cyclic form, both conformations and both alpha and beta isomers of the chair form are encompassed.
  • the terms “fermenting” and “fermentation” refer to a process in which microorganisms such as bacteria, yeast, and other small organisms metabolize one or more substances to produce the energy and chemicals needed to live and reproduce. This process of chemical reactions will produce some forms of by-product. Microorganisms are capable of generating a wide array of molecules as end points to fermentation. For example, carbon dioxide and ethanol are the by-products produced in brewing by yeast and pyruvate is converted into lactic acid in lactic acid fermentation. Fermentation is an ATP-generating process in which organic compounds act as both donors and acceptors of electrons, and it can take place in the absence of oxygen. Berg, M. Jeremy et al. Biochemistry chap. 16 (2002 ). As used herein, a "conventional fermentation” refers to a fermentation using only carbohydrate(s) as substrate(s).
  • feedstock is the raw material for fermentation. It may be a medium suitable for fermentation. It may further comprise fermentable substrates.
  • the substrates may include, but not limited to, carbohydrates and/or lactic acid, or mixtures thereof.
  • Other substances may also be present in the medium as needed.
  • NaOH, NH 4 OH, NaH 2 PO 3 , Na 2 HPO 3 , citric acid, HCl, NH 4 Cl may be added to adjust the pH value of the feedstock to a desired value, for example, pH 6, or to adjust other physical, chemical or physiological properties.
  • microorganism refers to a living organism too small to be seen with the naked eye, including bacteria, fungi, protozoans, algae, and viruses.
  • a strain of a bacterium may be a wild-type strain or a mutant strain.
  • Inoculum size refers to a ratio of the weight of cells (for example, immobilized cell beads) relative to the total volume of feedstock.
  • carbon yield is compared among fermentations using different feedstock and bacteria.
  • the possible chemical transformations are: Glucose(C 6 H 12 O 6 ) ⁇ 1 Butyric acid (C 4 H 8 O 2 ) + 2CO 2 + 2H 2 Glucose(C 6 H 12 OC 6 ) ⁇ 1 Butanol (C 4 H 10 O) + 2CO 2 + H 2 O
  • the measured carbon yield is often lower than the theoretical yield.
  • the gaseous product CO 2 is usually not a desired product in fermentation, and is usually considered “carbon loss.”
  • acetic acid and propionic acid are common products in fermentation and may be considered desired products in the "total carbon yield.”
  • total carbon yield For a fermentation which generates a significant amount of acetic acid and/or propionic acid, it is more appropriate to consider the "total carbon yield” to evaluate carbon retention efficiency.
  • the disclosure herein provides a novel method for producing butyric acid and butanol with improved carbon conversion and carbon yield, especially butyric carbon yield.
  • this is achieved by using lactic acid and at least one carbohydrate as substrates for the fermentation process.
  • a higher carbon yield especially butyric carbon yield, can be achieved, compared to the conventional fermentation using only carbohydrate(s) as substrate(s).
  • butyric carbon yield in the presently disclosed method may exceed about 66% (or 0.49 g/g), the theoretical butyric carbon yield in conventional fermentation, assuming the carbohydrate(s) proceed in the conventional fermentation mechanism.
  • the lactic acid fermentation proceeds through a different biochemical pathway, which does not involve the generation of CO 2 , that may explain why the total carbon yield of the methods disclosed herein exceeds the theoretical carbon yield of a traditional ABE pathway.
  • the present disclosure is directed to methods of producing butyric acid, comprising fermenting a feedstock with at least one butyric acid-producing bacterium to produce butyric acid, wherein the feedstock comprises lactic acid and at least one carbohydrate.
  • the disclosed method can increase the carbon yield of butyric acid by fermenting feedstock comprising lactic acid and at least one carbohydrate. Assuming butyric acid is the only fermentation product, the chemical equation of such conversion is as follows: 1 lactic acid (C 3 H 6 O 3 ) + 3H + + 3e - ⁇ 0.75 butyric acid (C 4 H 8 O 2 ) + 1.5 H 2 O
  • the method produces butyric acid by fermenting a feedstock containing lactic acid and at least one carbohydrate with at least one butyric acid-producing bacterium, for example, a Clostridium strain.
  • Clostridium strain include, but are not limited to C . tyrobutyricum, C. thermobutyricum, C. butyricum, C. populeti, C. cadaveros, C. cellobioparum, C . cochlearium, C. pasteurianum, C. roseum, C. rubrum, and C . sporogenes; such as C . tyrobutyricum, C. butyricum and C . beijerinckii.
  • Carbohydrates suitable for the disclosed method of producing butyric acid include, but are not limited to, fermentable monosaccharides, disaccharides, polysaccharides, and mixtures thereof.
  • the monosaccharide may be a pentose, or a hexose.
  • pentose include, but are not limited to, arabinose, lyxose, ribose, xylose, arabinose, ribulose, xylulose, and mixtures thereof; for example, xylose.
  • hexose examples include, but are not limited to, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, psicose, fructose sorbose, tagatose, and mixtures thereof; such as glucose and fructose; for example, glucose.
  • disaccharide examples include, but are not limited to, sucrose, lactulose, lactose, maltose, trehalose, cellobiose, and mixtures thereof; such as, lactose, sucrose and cellobiose; for example, lactose.
  • polysaccharides include, but not limited to, cyclodextrin, starches, glycogen, arabinoxylans, cellulose, guar gum, gum arabic, chitin and pectins.
  • the carbohydrates may be soluble carbohydrates obtained from treating biomass with an enzymatic saccharification process.
  • Suitable sources of biomass feedstock include agricultural residues such as corn stovers, corn cobs, and rice straw, and processing wastes such as corn fiber.
  • Suitable microorganisms for the saccharification include, but are not limited to, bacteria, yeasts, and filamentous fungi.
  • the weight ratio of lactic acid to the at least one carbohydrate in the feedstock ranges from about 1 to about 10, such as from about 1 to about 3, or from about 1 to about 1.5.
  • the carbon yield(s) refers to either “butyric carbon yield” or “total carbon yield,” as a general indication for carbon retention efficiency regardless whether butyric acid is the major product.
  • the method disclosed herein using lactic acid and at least one carbohydrate as feedstock provides good “butyric carbon yield” and “total carbon yield.”
  • the “butyric carbon yield” and the “total carbon yield” may be higher than 45%, higher than 50%, higher than 55%, higher than 60%, higher than 66%, higher than 67%, higher than 68%, higher than 70%, higher than 72%, higher than 73% or higher than 82%.
  • the butyric carbon yield and total carbon yield may range from 45% to 100%, from 50% to 100%, from 55% to 95%, from 60% to 90%, from 63% to 90%, from 66% to 90%, from 67% to 85%, or from 69% to 85%.
  • the butyric carbon yield and total carbon yield may vary for a number of reasons, for example, family and strains of the bacterium, whether the bacterium is immobilized or free suspension, carbohydrate(s), concentration and ratio of the lactic acid and carbohydrate(s), temperature, stirring speed of the fermentor, mode of fermentation (batch, continuous, etc.).
  • the fermentation process can provide a higher "butyric carbon yield” than a conventional fermentation using only carbohydrate(s) as the substrate(s). Higher “butyric carbon yield” may be achieved by (1) suppressing the generation of acetic acid, propionic acid and other products, and/or (2) decreasing CO 2 generation and minimizing carbon loss.
  • the butyric carbon yield may exceed 66%, the theoretical (maximum) butyric carbon yield.
  • the butyric carbon yield may be greater than 66%, greater than 67%, greater than 68%, greater than 70%, greater than 72%, greater than 73%, greater than 75% or greater than 82%, for example, from 66% to 90%, from 67% to 88%, or from 69% to 85%.
  • the lactic acid in the feedstock can be converted to butyric acid and other products with little or no production of CO 2 .
  • the butyric acid generated from lactic acid in a fermentation experiment using a combination of lactic acid and carbohydrate(s) as the substrates is calculated by subtracting the theoretical (maximum) butyric acid generated by the carbohydrate(s) from the total butyric acid produced.
  • the carbohydrates e.g., glucose
  • the lactic acid still generates good carbon yield compared to conventional fermentation using only carbohydrate(s) as substrate(s).
  • the lactic acid may generate a good carbon yield of greater than 45%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 80%, greater than 90%, and 100%.
  • the presently disclosed fermenting process can generate less CO 2 than a conventional fermentation.
  • the lactic acid in the feedstock may be converted to butyric acid with generation less than 1 molar equivalent of CO 2 , such as less than 0.5 molar equivalent of CO 2 .
  • the lactic acid in the feedstock is converted to butyric acid without generation of CO 2 .
  • the time period for which the method ferments can depend on a number of factors.
  • the fermentation time may depend on the strain of bacteria, concentration of the substrates, mode of operation, etc. Lactic acid may be consumed more rapidly, more slowly than the carbohydrate(s), or they may be consumed at about equal rate. In many circumstances, the butyric acid and other products are still generating after all the substrates are consumed.
  • the fermentation is allowed to continue until a substantial amount of the substrates is consumed, and/or no substantial increase in butyric acid is observed. For example, the amount of butyric acid usually stops increasing, or even decreases after a certain time due to product inhibition.
  • the duration of the fermentation process may last from about 10 to about 80 hours, for examples, from about 20 to about 80 hours, from about 30 to about 75 hours, from about 35 to about 70 hours, or from about 40 to about 55 hours.
  • the order of adding substrate(s) and the bacteria into the feedstock may be added in any order and is not restricted. Each substrate may be added multiple times before and during the fermentation commences. Lactic acid and carbohydrate(s) may be added into the feedstock sequentially and in any order.
  • the feedstock containing all substrates may be prepared before the inoculation.
  • One or more substrates may be added into the feedstock after inoculation repeatedly.
  • the feedstock may be separated from the bacteria (e.g., immobilized cell beads) and removed from the fermentor, and fresh feedstock is then added into the remaining bacteria in the fermentor to start a second fermentation. Such operation may be repeated as many times as appropriate.
  • the feedstock comprises lactic acid and at least one carbohydrate and the weight ratio of lactic acid to carbohydrate ranges from about 1 to about 10, such as ranging from about 1 to about 3, ranging from about 1 to about 1.5. In another example, the ratio may be about 1.
  • the butyric acid generated from the fermentation process may be further transformed to other organic molecules, such as butanol.
  • butyric acid may be hydrogenated to generate butanol.
  • the hydrogenating step may be a chemical hydrogenation, such as catalytic hydrogenation or transfer hydrogenation, in which the butyric acid is reduced by hydrogen gas or a hydrogen donor in the presence of a metal catalyst.
  • the hydrogenating step may be carried out using a butanol-producing microorganism, for example, a bacterium, or a solventogenesis phase of a bacterium, such as a Clostridium. Examples of Clostridium strains suitable for such transformation include, but are not limited, to C . acetobutylicum, C. beijerinckii, C.
  • the butyric acid obtained from the fermentation may further undergo esterification to generate butyrate ester, for example, in the presence of a catalyst and an alcohol, such as the methods described in U.S. Patent Application Publication No. 2010/0124773 .
  • the butyric acid may be extracted from the fermentation feedstock using an amine solvent.
  • Non-limiting examples of catalyst include lipase
  • non-limiting examples of alcohol include methanol, ethanol, propanol and butanol.
  • the catalyst maybe an enzyme, such as a lipase.
  • the esterification may take place without solvent if the alcohol is butanol, or with one or more solvents such as n-hexane, acetone, acetonitrile, cyclohexane, 2-octanol, Alamine 336, and a mixture thereof.
  • the resulting butyrate ester may be further reduced to butanol using chemical or biochemical methods.
  • a suitable apparatus for the disclosed fermentation process may be a free suspension system, such as the one illustrated in FIG. 1 .
  • the system includes a feedstock (26) with a cell suspension placed in a tank fermentor (10), which is equipped with a mechanical stirrer (12), a thermometer (14) and a pH meter (16), all connecting to or controlled by the integrated control station (30).
  • the integrated control station (30) controls the pH value of the fermentation by adding an acid solution (32) and a base solution (34) through tubings (70 and 72) to the acid and base inlets of the fermentor (18 and 20), the fermentation temperature by a thermostat (36), and the stirring speed (38).
  • An optional gas collecting device (50) may be attached to any fermentor described above to collect the gases, such as CO 2 and H 2 , generated during the fermentation through a gas outlet on the top of the fermentor (22) through a tubing (74). The gas generated may be analyzed by gas chromatography.
  • the method involves immobilizing the cells in beads of, e.g., polyvinyl alcohol (PVA) first.
  • the cell beads may be added to a bottle filled with a feedstock and the fermentation takes place in the sealed bottle.
  • the method uses immobilized cell beads in a stirred-tank reactor system, such as the one illustrated in FIG. 2 .
  • the system includes immobilized cell beads (24) with the feedstock (26) that is placed in the fermentor (10), which is equipped with the mechanical stirrer (12), the thermostat (14) and the pH meter (16), all connecting to or controlled by the integrated control station (30).
  • the integrated control station (30) controls the pH value of the fermentation by adding the acid solution (32) and the base solution (34) through the tubings (70 and 72) to the acid and base inlets of the fermentor (18 and 20), the fermentation temperature by the thermostat (36), and the stirring speed (38).
  • the optional gas collecting device (50) may be attached to any fermentor described above to collect the gases, such as CO 2 and Hz, generated during the fermentation through the gas outlet on the top of the fermentor (22) through the tubing (74).
  • the collected gas may be analyzed by gas chromatography.
  • the immobilized cell beads may be used in a recirculating pack-bed fermentation system comprising a fermentor and a pack-bed bioreactor, such as the system illustrated in FIG. 3 .
  • the system includes the feedstock (26) that is placed in the fermentor (10), which is equipped with the mechanical stirrer (12), the thermostat (14) and the pH meter (16), all connecting to or controlled by the integrated control station (30).
  • the integrated control station (30) controls the pH value of the fermentation by adding the acid solution (32) and the base solution (34) through tubings to the acid and base inlets of the fermentor (18 and 20), the fermentation temperature by the thermostat (36), and the stirring speed (38).
  • the fermentation system also includes a packed-bed bioreactor (60) containing immobilized cell beads (62) which is connected to the stirred-tank fermentor (10) through the recirculation loop (64).
  • the control station (30) also contains a pump (40) to transport the feedstock from the top of the fermentor through the recirculation loop (64) into the bottom of the pack-bed bioreactor.
  • the gas generated from the process is released through the gas outlets on the top of the fermentor and the bioreactor (22 and 66) to the optional gas collecting device (50) through the tubing (74).
  • the collected gas may be analyzed by gas chromatography.
  • Clostridium tyrobutyricum ATCC 25755
  • Clostridium butyricum IAM 19001
  • Clostridium beijerinckii ATCC 8260
  • BCRC Bioresource Collection and Research Center
  • the basal medium contained the following ingredients in per liter of deionized water: 5 g of yeast extract, 5 g of peptone, 3 g of ammonium sulphate, 1.5 g of KH 2 PO 4 , 0.6 g of MgSO 4 ⁇ 7H 2 O and 0.03 g of FeSO 4 ⁇ 7H 2 O. Wu et al., Biotechnology and Bioengineering 2003, 82(1), 93-102 .
  • the feedstock was prepared by adding appropriate substrate(s) to the basal medium. All media and feedstocks were sterilized by autoclaving at 121 °C, 15 psig, for 30 min prior to use.
  • a 5-L fermentor filled with 4-L of the basal medium containing glucose and lactic acid as the substrates was inoculated with about 300mL of a cell suspension prepared in serum bottles and then allowed to grow for 7 days until the cell concentration reached an optical density (OD 600 ) of about 5.
  • the cells were immobilized in phosphorylated PVA gel beads according to the method described in Chen, K., "Immobilization of microorganism with phosphorylated polyvinyl alcohol (PVA) gel.” Enzyme Microbiob. Technol. 1994, 16, 679-83 .
  • the cells were harvested by centrifugation at 6,500 rpm (KUBOTA, model 7780) for 10 min and suspended in an aqueous solution of PVA, about 9% (w/v), with a ratio of 20 g wet cell per liter of PVA solution.
  • the cell suspension and the PVA solution were thoroughly mixed, and the PVA-cell mixture was dropped into a saturated boric acid and sodium phosphate solution and gently stirred for 1-2 hr to form spherical beads.
  • the resulting beads of 3-4 mm diameter were rinsed with water.
  • Free cell density was analyzed by measuring the optical density of the cell suspension at a wavelength of 600 nm (OD 600 ) with a spectrophotometer (OPTIZEN, model 2120UV plus).
  • the system includes a 2-L tank fermentor, a pH meter connecting to the integrated control station, which controls the pH value of the fermentation by adding acid/base solution to the fermentor.
  • the carbon yield varies with the Clostridium strain used in the fermentation.
  • C . tyrobutyricum provides 56% butyric carbon yield overall, while lactic acid itself provides 48% carbon yield (assuming the conversion of glucose to butyric acid is at its theoretical yield, 66%), see FIG. 4 .
  • C . butyricum was used, a portion of lactic acid was utilized by the strain, although a large amount of lactic acid remained unchanged throughout the fermentation ( see FIG. 5 , top). That is, the specific strain does not convert lactic acid as efficiently as C . tyrobutyricum does, but the consumption of lactic acid is still achievable. The observation is consistent with the result of the fermentation using glucose as the sole substrate ( see FIG.
  • Example 2 the study investigated the effects of the lactic acid-carbohydrate combination on carbon yields in fermentation by immobilized C. tyrobutyricum beads in sealed bottles.
  • the feedstock was prepared using a modified basal medium, in which a phosphate buffer solution (consisting of 0.2M Na 2 HPO 4 and 0.2M NaH 2 PO 4 ) replaces distilled water.
  • the feedstock for the first experiment contained only 3.3 g/L glucose as the substrate.
  • the feedstock for the second experiment included 3.3 g/L glucose and 5.9 g/L L -lactic acid as the substrates
  • the feedstock for the third experiment included only 5.7 g/L L -lactic acid as the substrate.
  • the difference between total carbon yield and butyric carbon yield is greater than when a combination of glucose and L -lactic acid is used, because higher amounts of acetic acid and propionic acid are generated during the fermentation. See FIG. 7 .
  • the butyric carbon yield (61 %) is much higher than that of free cell fermentation (see Table 1, 36%), because the immobilized cell beads can be used repeatedly for a long period of time, which may cause cell adaptation to endure higher butyrate concentration, thereby resulting in a higher butyric carbon yield.
  • Example 3 the experiments studied the effects of bacteria on carbon yields of fermentation using an immobilized stirred-tank reactor system, illustrated in FIG. 2 .
  • the apparatus included a 2-L stirred tank fermentor equipped with a mechanical stirrer, a pH meter connected to an integrated control station, which controls the pH value of the fermentation by adding acid or base solution to the fermentation tank.
  • the fermentation commenced when the immobilized cell beads are added to the fermentation tank.
  • the gas generated during fermentation is collected through an outlet on the top of the fermentor and can be analyzed by gas chromatography.
  • the batch fermentation was performed in a stirred tank fermentor. After the fermentor was filled with the feedstock, containing glucose and L -lactic acid as substrates (concentrations shown in Table 3), anaerobiosis was reached by sparging the medium with N 2 . The pH of the medium was adjusted to 6.0 with 2 N NaOH before inoculation with 70 g of PVA-immobilized cell beads containing either C . tyrobutyricum or C . butyricum. The fermentation was carried out at 37°C, agitated at 600 rpm, and the pH was maintained at about 6 ⁇ 0.1. The reaction was allowed to continue until butyric acid ceased to generate due to product inhibition.
  • the CO 2 generated during the fermentation was monitored. As illustrated in the bottom graphs of FIG. 10 , the accumulated amount of CO 2 observed was less than 1.0 L (less than 0.5 molar equivalent) throughout the process, far less than the theoretical amount expected in a conventional fermentation using glucose alone. This suggests a different biosynthetic pathway which reduces the CO 2 production during the fermentation.
  • Example 4 the experiments studied repeated batch fermentation by reusing the same immobilized cell beads in a stirred tank reactor system, which is described in Example 3 and illustrated in FIG. 2 .
  • the batch fermentation of C . tyrobutyricum was performed in a 2-L stirred tank fermentor, and 1.0 L feedstock containing glucose (13 g/L) and L -lactic acid (15 g/L) was used in the first fermentation cycle. Anaerobiosis was reached by sparging the medium with N 2 . The pH of the medium was adjusted to 6.0 with 2 N NaOH before inoculation with 70 g of immobilized C . tyrobutyricum beads (inoculum size about 5% w/v). The experiment was carried out at 37°C, agitated at 600 rpm and the pH was maintained at around 6 ⁇ 0.1. Samples were taken at regular intervals for the analysis of free cell, substrate, and product concentrations.
  • the time course of the concentrations is illustrated in FIG. 11 .
  • the starting concentration of the substrates and the carbon yields are summarized below in Table 4.
  • the fermentation was continued until no more butyric acid was generated (about 70 hours after fermentation commenced).
  • the feedstock was removed, and the cell beads remained in the fermentor.
  • a butyric carbon yield of 65% was achieved and the butyric carbon yield for lactic acid was 64% assuming the glucose was converted with theoretical yield of 66%.
  • the horizontal dash line indicates the concentration of theoretical maximum butyric acid produced from glucose. Table 4.
  • Example 5 the experiment provided a fermentation process using immobilized C. tyrobutyricum beads.
  • the procedure and the fermentation system were substantially similar to those described in Example 3.
  • the batch fermentation was performed in a 2-L stirred tank fermentor. After the fermentor was filled with the feedstock containing glucose (15 g/L) and L -lactic acid (15 g/L), anaerobiosis was reached by sparging the medium with N 2 . The pH of the medium was adjusted to 6.0 with 2 N NaOH before inoculation with 70 g of PVA-immobilized cell beads containing C . tyrobutyricum (inoculum sizes about 5% w/v). The fermentation was carried out at 37°C, agitated at 600 rpm, and the pH was maintained at 6 ⁇ 0.1 by adding 2N NaOH solution. The reaction was allowed to continue until butyric acid ceased to be generated due to product inhibition.
  • the time course of the concentrations is illustrated in FIG. 12 , in which the horizontal dash line indicates the concentration of the theoretical maximum butyric acid produced from glucose.
  • the final butyric acid concentration was 17.2 g/L, and the amount of acetic acid in the product was very low.
  • the butyric carbon yield was 83%, far exceeding the theoretical yield of 66% from a conventional fermentation. Assuming glucose was converted with the theoretical carbon yield, the conversion from lactic acid to butyric acid was quantitative. That is, no carbon loss occurred through the generation of CO 2 .
  • the exceedingly high carbon yield in this example suggests an alternative biosynthetic pathway of butyric acid from lactic acid, which reduces the generation of CO 2 and increases the maximum carbon yield, compared to the fermentation without adding lactic acid.
  • Example 6 the study investigated the applicability of the fermentation process using carbohydrates other than glucose.
  • a packed-bed fermentation system used in the following experiments is illustrated in FIG. 3 .
  • the fermentation system included a 0.5-L packed-bed bioreactor connected to a 2-L stirred-tank fermentor through a recirculation loop powered by a pump in the integrated control station.
  • the integrated control station adjusts the pH value and temperature inside the fermentor.
  • a pump in the control station transports the feedstock from the top valve of the fermentor through the recirculation loop into the bottom of the pack-bed bioreactor.
  • the air generated from the process is released through the top of the bioreactor and fermentor to a gas collecting device.
  • the fermentation was operated under well-mixed conditions with controlled pH and temperature.
  • the carbon yields for xylose was the average of the two feedings. Good butyric carbon yields and total carbon yields were obtained in the fermentation process using a pentose such as xylose.
  • the experiment demonstrates that the combination of a carbohydrate other than glucose with lactic acid as substrates in fermentation exceeds those results anticipated under conventional fermentation using glucose as the only substrate, and in some instances may be deemed synergistic with the improvement of the butyric carbon yield.

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